There are a number of known methods available for peptide synthesis. A classical approach is liquid-phase peptide synthesis (LPPS), which has been a preferred method for producing large quantities of peptides. Another current and commonly used approach for peptide synthesis is solid-phase peptide synthesis (SPPS), wherein the growing peptide chain is covalently attached to a resin on a solid support, until cleaved from it once the desired length and sequence is achieved. In these methods reactive side chains of the incorporated amino acids need to be protected in order to avoid other reactions apart from the desired formation of new peptide bonds in the growing peptide. In addition, to avoid side reactions between the added amino acids, as well as incorporation of multiple amino acids in each step, the added amino acids are normally α-amino protected. The synthesis thus becomes one of repeated cycles of deprotection of the α-amine of a solid-phase attached peptide, followed by coupling to a single, α-amino protected amino acid unit.
Degarelix is a GnRH antagonist for use in the treatment of prostate cancer. Degarelix has an immediate onset of action and suppresses gonadotropins, testosterone, and prostate-specific antigen (PSA). Degarelix is a synthetic decapeptide of the formula Ac-D-2Nal-D-Phe(4Cl)-D-3 Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2.
The fifth amino acid moiety from the amino terminal of degarelix corresponds to the non-natural amino acid Aph(L-hor). Aph(L-Hor) stands for (L-hydroorotyl)-4-amino-phenylalanine. It is known in the art (Koedjikov, A. H. et. al., J. Chem. Soc. Perkin, Trans. 2, 1984, pages 1077-1081; Kaneti, J. et. al., Org. Biomol. Chem., 2004, pages 1098-1103) that, under basic conditions, compounds comprising a dihydrouracil moiety undergo rearrangement to compounds comprising a hydantoin moiety. The corresponding rearrangement of Aph/L-Hor) is illustrated below (upper left: dihydrouracil moiety N-4-(L-hydroorotylamino)-phenylalanine I, R=—CH2CHNH2COOH; lower right: hydantoin moiety II, N-4-[2-(5-hydantoyl)-acetyl)-phenylalanine).
In the rearrangement, the dihydrouracil moiety I is converted to a hydantoin moiety II. The L-Hor moiety of 4Aph(L-Hor) being of the dihydrouracil kind such rearrangement is expected to occur during a process of manufacture of degarelix in which basic conditions are employed. This was confirmed by the applicant by contacting peptide synthesis intermediates comprising α-amino group Fmoc-protected terminal 4Aph(Hor) with either NaOH or the organic base dicyclohexyl amine (DCHA). The deprotection product obtained was found to be contaminated by up to several % by weight of the corresponding hydantoin rearrangement product. In the synthesis of degarelix the intermediate Fmoc-4Aph(Hor)-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH-Resin thus can be expected to be partially rearranged to Fmoc-X-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH-Resin, X being 4-([2-(5-hydantoyl)]acetylamino)-phenylalanine when deprotected under basic conditions. Consequently, a degarelix product obtained via Fmoc-4Aph(Hor)-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH-Resin thus can be expected to be contaminated by a corresponding amount of Ac-D-2Nal-D-Phe(4Cl)-D-3 Pal-Ser-X-D-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2. Degarelix is the active ingredient of a drug for administration to humans. Therefore it must not be contaminated by any impurity exceeding 0.3% by weight of the product. Thus, in degarelix suited for human consumption the hydantoin by-product cannot be tolerated in an amount of more than 0.3 by weight. Since the hydantoin-moiety containing by-product is structurally very similar to degarelix, their separation is difficult. If attempted, separation is expected to result in substantial loss of product. Hence, in a process of manufacture of pharmaceutical-grade degarelix employing the protecting group Fmoc, basic conditions should be avoided.
The synthesis of degarelix is disclosed in U.S. Pat. No. 5,925,730A. The preferred α-amino protecting group in this synthesis and which has been used in all Examples is the tert-butyloxy-carbonyl group (Boc). In addition a wide range of other well-known protecting groups, such as the fluorenylmethyloxycarbonyl group (Fmoc) are disclosed for this purpose. An advantage with the Boc group is that α-amino groups protected by it can be deblocked under acidic conditions by standard treatment with trifluoroacetic acid (TFA). A disadvantage with TFA is its high human toxicity, which puts manufacturing personnel at risk. Another disadvantage with TFA is its environmental toxicity, which either makes it disposure costly or, if disposed improperly, contaminates the environment.